The long-term goal of this application is to improve our understanding of the mechanisms through which Notch signaling, a major cellular communication pathway, regulates mammalian development and stem cell-based maintenance of adult tissues, and to apply such insights to tissue engineering and disease abatement. Pathway activation occurs when ligand binding to any of the four vertebrate Notch receptors triggers proteolytic release of a signal "unit" (the Notch intracellular domain;NICD). All 4 NICDs engage in transcription by forming a complex with RBPjk, a DNA-binding protein. Since all NICDs bind to RBPjk with equal affinity, co-expression of Notch paralogs results in redundancy, thereby leading to robustness (insensitivity to loss of one allele). However, several observations suggest that Notch receptors are not always redundant when co-expressed, resulting in developmental syndromes when one copy of a specific mammalian Notch receptor is mutated despite the presence (and in some cases, activation) of other highly conserved Notch paralogs. In addition to this context-specific activity, we identified NICD paralog-specific activities in 3 tissues where Notch paralogs are individually expressed: T-cells, B-cell and endothelial cells. This application will use a multi-pronged approach aimed to determine the mechanistic basis for paralog-specific Notch activities in these tissues. Attaining a better understanding of how context affects Notch signaling output goes beyond the academic interest in the mechanism of Notch signaling, paving the way for novel, receptor- and tissue-specific treatment of developmental syndromes associated with Notch loss, receptor-specific inhibition in cancer, Notch receptor- specific contribution to the stem cell-niche interactions and selective activation of desired paralogs for organ engineering initiatives. PUBLIC HEALTH RELEVANCE: The Notch pathway constitutes a short-range communication channel used to regulate proliferation, stem cells and stem cell niche maintenance, cell fate acquisition, differentiation and cell death. In humans, two structurally similar receptors (Notch1 and 2) seem to work redundantly in many cells, but have unique properties in others. Loss of one Notch1 allele leads to aortic valve defects despite the presence of Notch2, and loss of one Notch2 allele leads to Alagille syndrome despite the presence of Notch1. Clues we obtained from the immune system suggest that part of the answer lies in the composition of the intracellular domain, perhaps permitting association with receptor specific partners. Our proposal aims to understand better how Notch1 and Notch2 work to produce unique cellular outcomes. Finding partners will allow us to target the pathway in a context specific manner. We therefore believe that the proposed research will improve our ability to manipulate Notch signaling where desired as a therapeutic key to populations with rare diseases and in stem cell based therapies.